JAEA-Review-2010-065.pdf:15.99MB - 日本原子力研究開発機構

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Behavior of Eu during Culture of Paramecium bursaria
with Yeast Cells Sorbing Eu
N. Kozai a) , T. Ohnuki a) , M. Kohka b) , T. Satoh b) , T. Kamiya b) and F. Esaka c)
a) Advanced Science Research Center, JAEA,
b) Department of Advanced Radiation Technology, TARRI, JAEA,
c) Division of Environment and Radiation Sciences, NSED, JAEA
It is known that activity of microorganisms such as
bacteria, algae, and yeasts has a great impact on the
geological migration of the radionuclides leached from the
radioactive waste forms buried underground. Retardation
of the radionuclide migration by adsorption and
mineralization on the cells is the most desirable function of
those microorganisms. It is also known that protozoa, who
prey those microorganisms, are found not only in surface
water and soil but also in deep ground water. Protozoa
defined as one-celled (unicellular) organisms control the
number of those bait-microorganisms. However, no
knowledge on the role of protozoa on the radionuclide
migration is available. The chemical forms of the
radionuclides sorbed on or taken up by those
bait-microorganisms may change during the process of
digestion and absorption by protozoa. Protozoa may take
up radionuclides directly from water. The objective of this
study is therefore to elucidate the role of protozoa in the
migration of radionuclides. This study selected paramecia
as model protozoa. Paramecia are the most common
species of protozoa in fresh waters and most extensively
used in research of behavior, heredity, and so on.
Depending on the species, paramecia are 70-350 µm length
and several tens of µm wide. This size is suitable for
micro-PIXE analysis considering the special resolution, less
than 1 µm, of the micro-PIXE analyzing system in the
TIARA facility.
In the first year of this study, uptake or sorption of metals
in aqueous solutions at pH 7 by Paramecium bursaria was
1)
investigated . It was found that Sr, Eu, and Pb were
hardly adsorbed on living cells of P. bursaria. It is very
interesting why Eu and Pb, which are very adsorptive to
bacterial cell surfaces, are not adsorbed on P. bursaria cells.
No evidences for mineralization of those metals on cell
surfaces of P. bursaria were also obtained. In the second
year of this study, we investigated behavior of Eu during
culture of P. bursaria in media containing yeast sorbing Eu.
Yeast, Saccharomyces cerevisiae, was used as a food source
and Eu(III) was used as simulant of trivalent actinides.
Yeast cells were precultured in a nutrient medium,
collected by centrifuge, and washed with an inorganic
aqueous medium containing 200 mg/L Ca(NO3) 2•4H2O, 20 mg/L MgSO4•7H2O, 0.8 mg/L Fe2(SO4) 3•nH2O, and
590 mg/L NaCl (Medium A) repeatedly. The yeast cells
were contacted with an aqueous solution containing 0.5 mM
Eu(OCOCH3) 3•nH2O for four days at 25 o C. After the
contact, the cells were separated from aqueous phase by
JAEA-Review 2010-065
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centrifuge and washed with Medium A repeatedly. Cells
of P. bursaria were precultured, collected, and washed with
Medium A repeatedly. Finally, P. bursaria cells were
statically cultured with the above prepared yeast cells in
flasks containing Medium A at 23 o C. The culture was
stirred only before periodic sampling of the P. bursaria cell
culture. The cells in the sampled culture were quickly
washed with Medium A and fixed with a fixative containing
4% glutaraldehyde and 60 mM sodium cacodylate. The
cells fixed were washed with purified water, dried on a
carbon foil in air at room temperature, and analyzed by
micro-PIXE.
A part of the Eu adsorbed on yeast cells was precipitated
as phosphate nano-particles on the cell surfaces and the rest
of the Eu seemed to be adsorbed on the cell membrane.
Soon after introducing of the Eu-adsorbing yeast cells Eu
concentrations in the aqueous phase increased up to about
1 µM but quickly decreased to an almost constant level, less
than 0.1 µM, after the second day of the culture. The
amount of Eu leached into the aqueous phase was less than
0.1% of the Eu on the yeast cells. As culture time
advances, membranous precipitates formed. These
membranous precipitates contained undigested and digested
yeast cells and dense membranous organic substance filling
gaps between those cells. At the end of culture, numerous
nano-particles of Eu phosphate were observed on digested
yeast cells in the membranous precipitates. These results
indicate that the Eu fixed on yeast cells mostly transitioned
into membranous precipitates through growth of P. bursaria
cells and thus suggest that Paramecium sp. do not impair
actinide-retardation action of bait-microorganisms.
Eu was detected by micro-PIXE for the P. bursaria cells
collected at inductive phase of growth (up to the first four
days of the culture). As culture time advances, Eu was not
detected for any cells. It is very interesting that Eu was
specifically concentrated in the P. bursaria cells at inductive
phase despite the fact that Eu was toxic to paramecium.
It seemed that the Eu spread throughout the cells at
inductive phase. This Eu is supposed to be in the interior
of the cells because the first year study revealed that
adsorption of Eu on cell surfaces of P. bursaria is very
unlikely. We will investigate intracellular distribution of
Eu concentrated in P. bursaria cells by 3D-micro-PIXE.
Reference
1) N. Kozai et al., JAEA Takasaki Ann. Rep. 2008 (2009)
31.